PREFI LEC: GENE MUTATIONS Flashcards
Permanent alteration in the DNA sequence that makes up a gene
GENE MUTATION
Causes of GENE MUTATION?
errors in DNA replication, exposure to various environmental factors (radiation, chemicals, or certain viruses)
Wide range of effects GENE MUTATION?
harmless, genetic disorders, development of diseases (cancer)
substitution of 1 nucleotide, will not change the amino acid sequence
Silent
change the amino acid sequence, but the replacement & the original amino acid have similar biochemical properties
Conservative
replacement of amino acid w/ a biochemically different amino acid
Nonconservative
terminates proteins prematurely when a nucleotide substitution produces a stop codon
Nonsense
addition/deletion of nucleotides in a DNA sequence disrupts the reading frame
Frameshift
purine replaces a purine or pyrimidine with a pyrimidine
Transition
purine replaces a pyrimidine or pyrimidine with a purine
Transversion
Used to directly analyze the change in protein structure/function Other uses:
Metabolic defects where several genes are involved in the disease phenotype
Detection of the actual protein/amino acid alterations
BIOCHEMICAL METHODS
WHAT ARE THE 5 BIOCHEMICAL METHODS
- Enzyme immunoassays 2. Immunohistochemistry 3. High-performance liquid chromatography (HPLC)
- Gas chromatography
- Mass spectrophotometry
Detects the presence of metabolites in the blood, urine, or other biological fluids
ENZYME IMMUNOASSAYS
Involve the use of specific antibodies or other ligands to detect the presence of the target molecules
ENZYME IMMUNOASSAYS
Widely used EIA
enzyme-linked immunosorbent assays (ELISA)
Longstanding method that allows detection of protein abnormalities in situ
IMMUNOHISTOCHEMISTRY (IHC)
Formalin-fixed paraffin-embedded tissue:
<5 micron slices (microtome)
Fixation can affect tissue antigens altering/covering some epitopes can be solved by antigen retrieval: enzyme digestion (proteinase K, chymotrypsin, pepsin, pronase) & heating tissue sections in water/buffer
Formalin-fixed paraffin-embedded tissue
Snap frozen tissue (in isopentane, at -160ºC):
5 to 15-micron slices (cryostat inside of a chamber held at 20ºC)
SNAP FROZEN TISSUE FIXATIVE
ACETONE
Sections are dried & stored frozen
SNAP FROZ2N TISSUE
Rehydration of the dried sections in of snap frozen tissue
phosphate buffered saline
2 Imaging/microscopic observation of antibody binding requires a signal from the antibody:
Fluorescent signal
Colorimetric signal
fluorescent molecules (fluorescein, Cy5, phycoerythrin)
Fluorescent signal
substrate solution is added, oxidized by the enzymes (horseradish peroxidase/alkaline phosphatase) most frequently used: red/ brown IHC staining
Colorimetric signal
Separation of molecules (NA & proteins) in solution through interaction with a solid support in the column
HIGH-PERFORMANCE LIQUID
CHROMATOGRAPHY (HPLC)
2 PHASE OF HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
mobile phase (solvent) & stationary phase (solid support)
increase resolution & lower separation time while using less solvent; faster flow rates (5 mL/min)
Ultra-HPLC (UHPLC)
Separation of vaporized sample through a column of inert carrier gas (mobile phase) & liquid (stationary phase) that differently adsorbs molecules
GAS CHROMATOGRAPHY (GC)
DETECTOR OF GAS CHROMATOGRAPHY (GC)
flame ionization detector
Used for detection of drugs & poisons & their metabolites in biological samples
May be coupled with MS to detect biomarkers of disease
GAS CHROMATOGRAPHY (GC)
Converts molecules to ions that can be moved in a magnetic field based on their charge & mass
MASS SPECTROMETRY
(MS)
2 ionization methods for large biomolecules (proteins) of MASS SPECTROMETRY (MS)
a. Electrospray ionization (ESI)
b. Matrix-assisted laser desorption/ionization (MALDI)
High MW molecules
Ionized molecules are accelerated at a fixed point & allowed to drift through the flight tube to the detector
MALDI-TOF (time-of-flight) spectrometry
Combined w/ TOF offers flexibility in the ID & quantification of peptides
Surface-enhanced laser desorption/ionization (SELDI)
Performed on a variety of specimen types: blood/buccal cells
DNA mutations from single-base pair changes to large chromosomal rearrangements can be detected
NUCLEIC ACID ANALYSES
simplified mutation detection (limiting specimens)
PCR amplification
most definitive method for detecting mutations
DNA sequencing
4 HYBRIDIZATION-BASED METHODS
Single-Strand Conformation Polymorphism (SSCP)
SEQUENCING (POLYMERIZATION) BASED METHODS
ENZYMATIC & CHEMICAL CLEAVAGE METHODS
OTHER METHODS
Based on the preference of DNA to exist in a double-stranded state
Absence of the complementary strand: nucleic acids form intrastrand duplexes, 3D structure (conformer)
Shape: kinks, loops, bubbles, tail
Single-Strand Conformation Polymorphism (SSCP)
Different from the normal sequence (control) conformers
presence of gene mutation
Detected by silver stain, radioactivity, or fluorescence
Bands/peak patterns
Utilizes differences in the Tm of short sequences (20 bases) w/ 1 or 2 mismatches & those w/ no mismatches
Synthetic ss-probes (labeled) w/ normal/mutant target DNA sequence (immobilized) in a solution
At specific annealing temperatures & conditions (stringency)
a. Probe will not bind to a near complementary target sequence w/ 1 or 2 mismatched bases
b. Probe w/ perfect complementary sequence, will bind
Allele-Specific Oligomer Hybridization
(ASO)
Method of analyzing the dissociation of dsDNA during the heating cycles
PCR amplicons in the presence of a DNA-specific fluorescent dye (EtBr, SYBR green, LC green) are heated (0.3ºC/sec)
Rise in the temperature, DNA duplexes begin to separate into single strands, losing the dye
Black line
targets w/ different mismatches to the hybridization probe
Melt Curve Analysis (MCA)
overlaying peaks at expected Tm
Specimen w/ identical sequence
2 or more peaks at different temperatures
Specimen w/ different sequence
High specificity Uses fluorescent resonance energy transfer (FRET) probes
Raise in temperature, probes dissociate at specific Tm donor no longer close to the acceptor fluorescence drops
High-resolution melt-curve analysis (HRMCA)
Tm lower than that of the probe & its perfect complement, or sequence difference between probe reference sequence & the test sequence
High-resolution melt-curve analysis (HRMCA) Indication of mutation
Formed when single strands that are not completely complementary hybridize to 1 another
Heteroduplexes
Can be resolved through polyacrylamide/agarose gel electrophoresis:
presence of bands different from a homozygous reference control is indicative of mutation
Test DNA is fragmented by treatment with DNase before binding to the complementary probes on the array
High-density oligonucleotide arrays
2 TYPES OF Hybridization formats
Standard tiling
Redundant tiling
base substitution is always in the 12th position from the 3’ end of the probe
Standard tiling
same mutation is placed at different positions in the probe *Mutations are identified as indicated by which probes are bound
Redundant tiling
uses sets of color-coded polystyrene beads in suspension as the solid matrix
Bead-array technology
presence/absence of a mutation/polymorphism
Combination of bead color & test label
Detect point mutations & other SNPs Primer 3’ end falls on the nucleotide to be analyzed
Must match the template perfectly to be extended by Taq polymerase
Presence/absence of the product = presence/absence of the mutation
Sequence-Specific (Primer) PCR (SSP-PCR)
Thermal cyclers w/ fluorescent detection
RT-PCR, using 2 probes labeled 3’ quencher molecules & different fluors on the 5’ ends (complementary to either normal/mutant sequence)
Presence of corresponding fluorescent signal indicates whether the test sequence is normal/mutant
Allelic Discrimination with
Fluorogenic Probes
Can detect sequence alterations Mutation changes the structure of a restriction enzyme target site/changes the size of a fragment
PCR-RFLP is used
Restriction Fragment Length
Polymorphisms (RFLPs)
Heteroduplex analysis using duplex RNA
T7 or SP6 phage RNA polymerase Detection of mutation: heteroduplexes form between normal & mutant
transcripts
targets for cleavage by RNase enzymes (E. coli RNase & Aspergillus RNase T1)
Remaining dsRNA fragments can then be separated by agarose gel electrophoresis
Nonisotopic RNase Cleavage Assay
(NIRCA)
Based on the characteristic enzymatic activity of cleavase
Premixed reagents (including cleavase) + standard 96 well-plate + test specimens + controls
Cleavage Assay
recognizes the structure formed by hybridization of the normal/mutant probes to the test sequences
Cleavase
Combination of methods to increase sensitivity & detection (RFLP with modified primers)
other metjods
Array-based methods & massive parallel sequencing methods provide specific multiplex detection & sensitivity required for clinical applications
other methods
